PROJECT SUMMARY The microbiome is an essential component of health, and changes in the microbiome can lead to various diseases including diabetes, neurodegenerative diseases, inflammatory bowel disease, obesity, and cancer. This interaction between the microbiota in the gut and various neurodegenerative diseases, including central nervous system (CNS) tumors, is an area of critical importance. Recent studies have demonstrated the composition of the gut microbiome influences the efficacy of cancer immunotherapies. The immune checkpoint inhibitor anti-PD-1 demonstrates remarkable results in some patients with melanoma and lung cancer. Although anti-PD-1 works well in glioblastoma (GBM) pre-clinical mouse models, this therapy has not demonstrated similar efficacy in human GBM patient clinical trials. To date, all GBM pre-clinical studies have been done in mouse models with mouse gut microbiomes. There are significant differences between mouse and human microbial gut compositions, and some studies have found that 85% of gut bacteria found in laboratory mice are not found in humans. Surprisingly, the role of human microbiota in CNS tumors such as GBM has never been evaluated. Therefore, we have generated a unique humanized microbiome model in which mice have been colonized by human donor microbes (two separate healthy control donors-HuM1 and HuM2) to determine the effect of the human gut microbiome and response to therapy in pre-clinical models of GBM. Most surprisingly, we have found that one humanized line, HuM1, is resistant to anti-PD1, while the other line, HuM2, is responsive. These mice are genetically identical and only differ in gut microbiomes. The goals of this proposal are to 1) expand and extensively characterize our humanized microbiome mouse model, and 2) employ this novel mouse model to a CNS disease (GBM). Specific Aim 1: Extensive Characterization of Humanized Microbiome Mouse Model. We propose to generate an additional 4 humanized microbiome lines (with 4 different human donors) and more accurately identify individual microbiome compositions (metagenomic sequencing to identify species level identification of the microbes), immune cell subsets and phenotypes (both periphery and brain), and circulating metabolites (metabolomics). Specific Aim 2: Assess CNS Tumor Growth and Response to Therapy in the Humanized Microbiome Mouse Model. We will utilize our humanized microbiome mice to more accurately understand individual responses to tumor growth and pre-clinical drug screening (immunotherapy), and assess the responder microbiome as a potential therapy via fecal microbial transplant (FMT). The development and characterization of our novel humanized microbiome mouse model system has high impact for the neuroscience community. In future studies, these mice can be utilized as pre-clinical models of disease including Parkinson?s Disease, Multiple Sclerosis, autism and depression.